Image-based sample analytical measurement techniques have been limited by the number of molecular measurements that can be observed simultaneously (the extent of multiplexing) in a single sample, for example a tissue specimen. This has, so far, constrained this type of analytical approaches from large-scale ‘-omics’ use when compared to other highly multiplexed technologies, such as single-cell sequencing or mass cytometry. As a consequence, essential spatial details, which currently only image-based approaches can reveal, are being missed. Using immunofluorescence rather than classical immunohistochemistry can partly overcome this issue, but measurements are still limited to a maximum of 4-5 simultaneous molecular readouts. The major limitation of image-based multiplexed sample analytical measurements is the separation of distinct signals in a single specimen without cross talk between signals. In case of fluorescence imaging, for example, overlap of spectra prevents a clear separation of the emitted signals in a highly multiplexed multicolor labeling experiment. In addition, fluorophores may exhibit self-quenching behavior at high labeling densities, further limiting the simultaneous application of multiple labels. Another constraint on the multiplexing ability of immuno-based approaches is the requirement that each primary antibody has to be derived from different animal species to ensure specific amplification and detection with secondary antibodies. This could in principle be overcome by direct immunofluorescence, in other words labelling primary antibodies directly, but this approach gives rise to other problems, such as a decreased specificity and a lower signal output due to a lack of amplification.
Multiple molecular readouts using immunofluorescence have been achieved using an antibody mixing method shown in WO 2007/047450. While this method has the advantages of being image-based, applicable to tissue sections and able to be utilized in environments containing non-specific nucleases, the maximum number of simultaneous detections is limited. The inclusion of quantifiable reference standards into the measurement process such as described in WO 2008/005464, while increasing the precision in quantification immunohistochemistry read-outs in certain applications such as semi-quantitative scoring of biomarker proteins, e.g. as applied to semi-quantitative scoring of Human Epidermal Growth Factor 2 (HER2) expression in breast cancer tissues, may further limit the possibility of multiple simultaneous readouts because of dividing the output signals into specific bands.
Sample multiplexing with in situ imaging can be achieved by carrying out spectral multiplexing which comprises applying different stains on the same sample and extracting individual stain images from the imaging results as described in EP 1131631. The technique involves collecting spectral data from each pixel of the sample, computationally generating a spectrum that would have resulted from each individual staining and showing the individual results in a corrected colour spectrum. While allowing for multiple marker quantification, the device performance is inversely proportional to the number of parallel stains because of possible crosstalk between each signal.
Recent advances in immunostaining technologies are highly promising with regard to overcoming the above-mentioned limitations. These technologies make use of multi-cycle in situ imaging, which involves dye-inactivation and/or antibody elution after a usual staining/imaging step to enable additional rounds of staining and imaging.
Those approaches include chemical inactivation of fluorescent dyes after each image acquisition (Gerdes et al., 2013, PNAS, 110(29), 11982-11987), non-destructive dissociation of the antibody-antigen bonds for successive staining cycles by sequentially using a tailor-made acidified peimanganate solution (WO 2010/115089), successive antibody elution with various different buffers (Pirici et al., 2009, J. Histochem. Cytochem., 57(6), 567-575), successive cycles of peptide probe contact and denaturation at high temperature for sequential multi-target detection (WO 2009/117140), iterative staining and imaging cycles using a combination of denaturation and elution techniques (Wahlby et al., 2002, Cytometry, 47(1), 32-41), multiple sequential staining cycles using bleaching before each restaining step (Friedenberger, 2007, Nature Protocols, 2, 2285-2294), use of bioconjugated quantum dots as biological labels for multiplexed profiling of molecular biomarkers (Schubert et. al., 2006, Nature Biotechnology 24, 1270-1278), use of water soluble polymers forming bonds with multiple target molecules of interest (Xing, 2007, Nature Protocols 2, 1152 1165).
All those multi-cycle in situ imaging approaches are iterative and present the advantages of allowing subsequent utilization of primary antibodies raised in the same species as well as the same chemical reagent or fluorophore for different molecular targets and to theoretically enable identifying an unlimited number of different targets on the same tissue section.
Although promising, translation of multi-cycle in situ imaging technologies to high-throughput, multiplexed molecular profiling of samples such as for example tumour sections is not straightforward. First, long incubation and washing cycles (usually up to several hours) result in extremely long total protocol durations that cause degradation of tissue antigens under fluctuating ambient conditions. Second, repeated mounting/demounting of imaging of sample coverslips steps further deteriorate tissue integrity. Therefore, such manual handling of cycles affects reproducibility and practically impedes reliable molecular profiling of tissue specimens at high-throughput and renders the use of multi-cycle technologies impractical in applications like diagnostic purposes which require high throughput, reliability and relative low-cost implementation characteristics.
A further limitation of the existing methods available for in situ imaging of samples also originates from large-area imaging requirement for realizing multi-cycle assays. Since the specimens are removed from imaging systems to realize manual processing in between each cycle and following the manual processing, the specimens are restored back to imaging systems for a large-area imaging subsequent molecular marker and the images obtained from the specimen at all cycles are overlaid, subtle differences of specimen positioning on the imaging systems at each cycle introduce errors on localization of molecular signals throughout the specimen. This hinders the true localization of molecular signals, in particular those of subcellular features that can only be observed with a high resolution or super-resolution microscopy systems.
Vertical microfluidic systems have also been introduced as a possible tool to be used in immunoassays or genetic analysis. A microfluidic probe which is made up of a wide chamber and vertical access holes has been developed to stain small-area spots on a sample (WO 2014/001935). The dimensions of the stained area in each cycle are at the order of 100 μm. So, it is necessary to scan the sample surface with a number of staining steps to obtain a larger image. The issues of possible localization errors and analysis time increase resulting from the scanning process are also present in this method.
An open-top microfluidic device to facilitate easier transition between sequential staining and imaging steps has been recently presented (WO 2014/035917). The method aims to overcome the several disadvantages of having to de-coverslip the sample between each successive run such as extra time consumption, tissue loss and slide-to-slide image variation by eliminating the need for this step, while it does not address the imaging area and process time requirements.
Finally, it has been observed that in situ imaging involving sequential and repeated fluorophore exposure of the sample leads to induced damages on the sample. In particular, fluorescently labelled antibodies usually get cross-linked to the sample or tissue during imaging and cannot be removed from sample or tissue afterwards, which further limits the use of in situ imaging by cycle multiplexing.
Therefore, there is a need for new techniques, instrumentation and tools for in situ imaging of samples by cycle multiplexing which would allow multi-molecular read-outs on the same sample with high-throughput, high sensitivity, reliability and precision regarding the true localization of the molecular signals, notably for applications in the fields of diagnostics or treatment course monitoring in which the demand is currently considerably expanding.